Detection of analytes in a dual-mediator electrochemical test strip

ABSTRACT

Determination of an analyte such as glucose in a sample is done making use of a plurality of electron transfer reagents, for example two electron transfer reagents, that work together to transfer electrons between the enzyme and the electrodes. The first electron transfer agent is a mediator that interacts with the enzyme after it has acted on the analyte to regenerate enzyme in its active form. The second electron transfer agent is a shuttle that interacts with the electrodes and optionally the mediator. The oxidation and reduction of the shuttle serves as the major source of current that is measured as an indication of analyte.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/805,918, filed Jun. 27, 2007, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

This application relates to the electrochemical detection of analytes ina test sample. The invention is particularly applicable to the detectionof glucose in a sample of blood or other biological fluid using adisposable electrochemical test strip.

FIG. 1 shows a schematic representation of a conventional enzymebiosensor for the electrochemical detection of glucose. The biosensorhas a working electrode 10 and counter electrode 11 in a circuit with avoltage source 12. The sample is disposed between the electrodes 10,11to complete the circuit. When glucose is present, glucose oxidase, GOXoxpresent in the sensor oxidizes the glucose to gluconolactone and reducedenzyme, GOXred is formed. An oxidized mediator, MEDox, present in thesensor restores the enzyme to the active oxidized form, GOXox, andgenerates reduced mediator, MEDred. The applied voltage in the circuitis selected such that reduced mediator is oxidized at electrode 11 andthat oxidized mediator, MEDox, is reduced at electrode 10. Current flowwithin the sensor results from the oxidation and reduction of themediator at the electrodes, and this current flow is frequently measuredto assess the amount of glucose in the sample. The biosensor shown inFIG. 1 can be modified for use with other analytes, for example byselection of a redox enzyme (oxidase, dehydrogenase etc.) with differentspecificity appropriate for the other analyte.

Numerous mediators have been disclosed for use in biosensors of the typeshown in FIG. 1. In general, suitable mediators are ferricyanide,metallocene compounds such as ferrocene, quinones, phenazinium salts,redox indicator DCPIP, and bipyridyl-substituted osmium compounds. See,for example, U.S. Pat. Nos. 5,589,32, 6,338,790 which are incorporatedherein by reference. In selecting a mediator to use with a particularanalyte, several factors are generally relevant. For example, in thecase of glucose, the mediator is selected to have a redox potential thatallows it to regenerate the enzyme, glucose oxidase, from the reduced tothe oxidized state. In addition, if the kinetics of the reaction withthe enzyme are slow, the reduced enzyme may also react with oxygenpresent in this sample, leading to errors as a result in differences inhematocrit and blood pO₂. (FIG. 2) Thus, it is also desirable to havefast kinetics for the enzyme-mediator reaction. It is frequently thecase, however, that compounds that meet the desired criteria for redoxpotential and kinetics are poorly soluble in aqueous solutions, such asblood. This means that the maximum concentration of mediator is limited,and as a result that the maximum amount of signal that can be generatedis limited.

SUMMARY OF THE INVENTION

The present invention provides for improved determination of an analytesuch as glucose in a sample by making use of a plurality of electrontransfer reagents, for example two electron transfer reagents, that worktogether to transfer electrons between the enzyme and the electrodes. Afirst electron transfer agent is a mediator that interacts with theenzyme after it has acted on the analyte to regenerate enzyme in itsactive form. The second electron transfer agent is a shuttle thatinteracts with the electrodes and optionally the mediator. The oxidationand reduction of the shuttle serves as the major source of current.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of an enzyme biosensor inaccordance with the prior art.

FIG. 2 shows a schematic representation of an enzyme biosensor inaccordance with the prior art with oxygen reaction with reduced enzymein competition with the mediator.

FIG. 3 shows a schematic representation of an enzyme biosensor inaccordance with an embodiment of the invention.

FIG. 4 shows a schematic representation of an enzyme biosensor inaccordance with an embodiment of the invention.

FIG. 5 shows a possible relationship between E⁰ values for the shuttleand mediator.

FIG. 6 shows another possible relationship between E⁰ values for theshuttle and mediator.

FIG. 7 shows a schematic representation of an enzyme biosensor inaccordance with an embodiment of the invention.

FIG. 8 shows a schematic representation of an enzyme biosensor inaccordance with an embodiment of the invention.

FIG. 9 shows experimental results using a dual mediator system of theinvention as compared to a single mediator system.

DETAILED DESCRIPTION OF THE INVENTION

In the present application and claims, the following definitions areapplicable:

“analyte” refers to the material of interest in a sample. The analytemay be, for example, a chemical of biological significance such asglucose, lactate, cholesterol, fructose, alcohol, amino acids, creatineand creatinine.

“detection of analyte in a sample” refers to the qualitative,semi-quantitative or quantitative determination of whether an analyte ispresent in a sample at detectable levels. Qualitative analysis providesmerely a yes/no answer as to whether there is detection.Semi-quantitative provides an indication of amount, but with highgranularity, for example as presented through a series of lights wherethe number of lights illuminated indicates the range into which a valuefalls. Quantitative analysis provides a numerical value for the amountof analyte in the measured sample.

“electron transfer reagent” refers to a compound other than a redoxenzyme that receives and donates electrons to and/or from anotherchemical species or to and/or from an electrode. The mediator compoundand the shuttle compound of the present invention are both electrontransfer reagents.

“mediator compound” refers to an electron transfer reagent that in usereacts with the inactive enzyme to regenerate active enzyme. Themediator compound may also transfer electrons to or from the electrodesalthough this is not required. In some embodiments, the mediatorcompound does not transfer electrons directly to or from the shuttlecompound. In other embodiments, electron transfer between mediator andshuttle in solution can happen and in other embodiments electrontransfer between mediator and shuttle is the most significant method oftransferring electrons from the mediator

“redox enzyme” refers to an enzyme that catalyzes oxidation or reductionof the analyte to be determined. Exemplary redox enzymes are oxidasessuch as glucose oxidase and dehydrogenases such as glucose dehydrogenaseor lactate dehydrogenase. The redox enzyme has an active form prior tointeraction with the analyte, and an inactive form. In the case of anoxidase, for example, the active form is oxidized and the inactive formis reduced.

“sample” refers to the material that is placed in the sample chamber toperform a determination for analyte. The sample is applied as a liquid,although samples that are not liquid in the first instance can becombined with a liquid to produce a sample for application. In general,the sample is a biological fluid such as blood, serum, urine, saliva orsputum.

“shuttle compound” refers to an electron transfer reagent that in usetransfers electrons to and from the electrodes. In some embodiments ofthe invention, the shuttle is reduced by transfer of an electron fromone electrode and oxidized by transfer of an electron to the otherelectrode. In other embodiments, the shuttle also can be reduced (oroxidized) by transfer of an electron from the reduced mediator (or tothe oxidized mediator). In this instance, in one preferred embodiment,the mediator itself does not react at the electrodes.

Theory of the Invention

The present invention utilizes a dual system of a mediator and a shuttlecompound to separate the characteristics of an “ideal” mediator into twoentities, where the overall result can come closer to ideality. Themediator in the present invention has an appropriate E^(o) value basedon the redox potential of the enzyme to be regenerated. In the case ofglucose oxidase, this is a value of less than 150 mV. For other enzymes,the value may vary somewhat. Second, the mediator has a fast rate oftransfer from the enzyme or an enzyme-coenzyme complex. “Fast” refers tothe rate of transfer relative to the rate of reaction of the reducedenzyme with oxygen. This rate is dependent on the specific enzyme.However, in general, it is desirable to have the rate of electrontransfer to the mediator be at least 10 times the rate of electrontransfer to oxygen, preferably at least 100 times, and even morepreferably at least 1000 times.

The shuttle compound used in the present invention is selected such thatit has slow or no reaction with oxygen. This reaction with oxygen isbased on the presence of at most minimal reaction with oxygen during theduration of the test. Slow means that there is some observable reactionof reduced shuttle with oxygen in this time period, but that the amountdoes not change the measured signal by more than 10%. No reaction meansthat there is no detectable oxygen-dependent variation in signal overthe duration of the test.

The shuttle compound is also “highly soluble.” In this context, the term“highly soluble” refers to solubility relative to the mediator employedin the sensor. This allows the concentration of shuttle to be in excessover the concentration of the mediator, for example more than 10 timesthe mediator concentration, preferably more than 50 times the mediatorconcentration, and more preferably more than 100 times the mediatorconcentration.

FIG. 3 shows the co-operation of the mediator and shuttle in accordancewith one embodiment of the invention. In this embodiment of theinvention, mediator interacts with the enzyme glucose oxidase and isoxidized/reduced at the electrodes 10, 11 in a conventional manner. Theshuttle compound is also oxidized/reduced at the electrodes 10, 11, butthere is no direct interaction between the mediator and the shuttle.Faradaic reaction of the shuttle is initiated by the passing of anelectron from the reduced mediator to the positive electrode 12, and thecompletion of the circuit by shuttle (ox) accepting an electron from thenegative electrode 11. Faradaic reaction of the mediator may also occurand the observed current is no different when it does. However, thegreater concentration of the shuttle will make this a lesser componentof the current.

FIG. 4 shows a schematic representation of a dual mediator sensor inaccordance with another embodiment of the invention. The sensor depictedin FIG. 4 is similar to the sensor of FIG. 3 except that in this casethere is no Faradaic reaction of the mediator at the negative electrode11.

The dual-mediator sensors as depicted in FIGS. 1 and 2 offer severaladvantages over conventional sensors because of the separation ofcharacteristics. These may include:

(1) The applied potential required to turn over the mediator (<150 mV)does not drive other redox reactions.

(2) The mediator can be chosen such that it has a fast rate of electrontransfer from the reduced enzyme, thereby limiting the loss of signaldue to oxygen reacting with the reduced enzyme, without worrying aboutalso solving the solubility issues.

(3) The shuttle reacts slowly, or not at all, with O2, so there isminimal or no loss of signal caused by oxidation of the reduced shuttleby O2.

(4) Contrary to the mediator, which is often limited to a lowconcentration by solubility or economic reasons, the shuttle is highlysoluble, so that Faradaic reaction of the shuttle is not limited byavailability of the molecule in solution.

(5) The shuttle has a fast rate of Faradaic reaction.

(6) The shuttle is inexpensive, so it is included in great excess overthe mediator, ensuring that a counter reaction is available and that theconstant current due to Faradaic reaction of the shuttle is maintained.

As is apparent from consideration of FIGS. 3 and 4, the potentialrequired to oxidize/reduce the mediator and shuttle may be different.This difference can be tuned through the selection of the mediator andshuttle to provide for the desired form of operation in the sensor. FIG.5 illustrates the case where the E⁰ of the mediator is greater than theE⁰ of the shuttle, i.e. E^(o) _(med)>E^(o) _(shut). The followingobservations can be made with respect to this situation. If V_(app) (theapplied potential) >E^(o) _(med), then both the mediator reaction andthe shuttle reaction proceed and measurements can be taken as per usual.If E^(o) _(shut)<V_(app)<E^(o) _(med), the shuttle reaction proceeds,but the mediator reaction does not, i.e. all of the mediator in solutionis trapped in the reduced state.

Since all of the mediator is trapped in the reduced state, the enzymereaction can be stopped, (assuming there is no oxygen to turn over theenzyme) by controlling the potential. This is a unique advantage sincewe can then selectively stop the enzyme reaction as some particularpoint in time before proceeding with a measurement. Non-limitingexamples of when this might be useful are:

(1) If the sensor's ambient temperature falls outside a range and it isdesirable to wait until the conditions become suitable for a measurement

(2) If there is excessive ambient noise (e.g. RF, vibration, etc) and itis desirable to wait until the noise subsides before making ameasurement.

(3) If there is insufficient sample in the test strip and it isdesirable to wait until the chamber is fully filled before continuingwith a measurement.

FIG. 6 shows an alternative selection of mediator and shuttle in whichE⁰ _(shut)>E⁰ _(med). In this case, V_(app) (the applied potential) mustbe greater than E^(o) _(shut) in order to ensure that both the mediatorreaction and the shuttle reaction proceed. It is advantageous to haveE^(o) _(shut)<150 mV, so that the applied potential required to turnover all reactions does not drive other redox reactions. This isimportant, since the blood contains several other redox active species(e.g., ascorbate, proteins, metabolites, etc.), which may contribute tothe electrochemical current, resulting in spurious blood glucosereadings.

FIG. 7 shows a schematic representation of a further embodiment of theinvention. In this embodiment, there is direct reaction between themediator and the shuttle, and the mediator does not significantlyundergo Faradaic reaction at the electrode. FIG. 8 shows a similarembodiment in which the reaction of the mediator with the shuttle iscoupled through a redox catalyst. This redox catalyst is a thirdelectron transfer agent.

In the foregoing discussion, the E⁰ of the mediator is referred to. Itshould be appreciated, however, that specific knowledge of absolute E⁰values is not required, and that what is important is the relativevalues for the materials being utilized as mediator and shuttle. Thiscan easily be obtained experimentally at the conditions found in thetest strip (pH, temperature etc) without requiring the more detailedmeasurements to determine an E⁰ value under standardized conditions.

Sensor of the Invention

According to one aspect, the present invention provides a sensor for thedetection of an analyte in a sample. The sensor of the present inventioncomprises:

(a) a support portion defining a sample chamber;

(b) a working and a counter electrode disposed within the samplechamber; and

(c) a reagent composition disposed in the sample chamber, said reagentcomposition comprising (i) an active redox enzyme effective to oxidizeor reduce the analyte, (ii) a mediator compound, and (iii) a shuttlecompound. In certain preferred embodiments, the sensor is intended forsingle use, and is used in combination with a reusable device or meterthat provides the analysis electronics and display or othercommunications means to convey a test result to the user. The sensormay, however, be part of an integrated device that includes both therecited elements and the electronics and display/communications means.

Sensors for use in the detection of glucose and other analyte speciesare known, and the structures of these sensors are representative of anduseable as the support portion and the electrodes of the sensor of thepresent invention. Specific non-limiting examples of support andelectrode configurations are found in U.S. Pat. Nos. 5,120,420,5,437,999, 5,958,199, 6,287,451, and 6,576,101 and US Patent PublicationNo. 2005-0258035, which are incorporated herein by reference.

The reagent composition disposed in the sample chamber contains anactive redox enzyme effective to oxidize or reduce the analyte. As willbe apparent, the specific enzyme is selected based on its specificityfor the analyte. Non-limiting examples of suitable enzymes includeglucose oxidase, fructose dehydrogenase, glucose dehydrogenase, alcoholoxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, andamino acid oxidases.

The general characteristics of compounds useful as mediators in thecompositions of the invention are set forth above. Specific,non-limiting examples of suitable mediators include osmium-containingmediators such as [Os(MeBpy)₂(Im)₂]^(2+/3+). The solubility of the PF₆salt of this complex is less than 1 mM and it has a redox potentialE^(o)=140-154 mV. The second order rate constant of electron transferfrom GOX to the 3+ mediator is approximately 4.0×10⁵ M⁻¹s⁻¹. Anotherspecific mediator is [Os(Mebpy)2Pic]^(+/2+). Other examples of goodmediators (which may not have good solubility) are: Ferrocenes and othermetallocenes in general, with various derivatizations (especiallysulfonation); Metal compounds (especially, but not limited to, Ru, Os,Fe, and Co) containing ligands of the following types, which may bederivatized with substituent groups to enhance solubility, tune redoxpotential and ligating abilities, or for other reasons: bipyridyl,phenanthroline, imidazole, thiolene/thiolate/thioether/sulfide,porphyrins, pyrrole/pyrrazole/thiazole/diazole/triazole, picolinate,carboxylate, oxo, quinone; Metal clusters (i.e. more than one metal inthe compound).

Non-limiting examples of suitable shuttles include:

Metal complexes (especially, but not limited to, Ru, Os, Fe, and Co) ofmonodentate ligands, including, but not limited to: hydrates/hydroxo,aminates, acetates, thiolates, halides, thiocyanates, cyanides,especially ferricyanide;

Metal complexes (especially, but not limited to, Ru, Os, Fe, and Co) ofmultidentate ligands (which may be derivatized to enhance solubility,tune redox potential and ligating abilities, or for other reasons),including, but not limited to: Aminoacetates, EDTA(Ethylenediaminetetraacetic acid), especially Fe(III)-EDTA, Ru(III)-EDTAand CO(III)-EDTA, HEDTra (hydroxyethylethylenediamine triacetic acid),NTA (Nitrilotriacetic acid), ADA (β-alaninediacetic acid), MGDA(methyleneglycine diacetic acid), IDS (iminodisuccinate), GLUDA(glutamate N,N′-bisdiacetic acid), EDDS (ethylenediamine disuccinicacid) DTPA (Diethylenetriaminepentaacetic acid);

-   -   Polyethers, for example cryptates and/or encapsulating ligands        and crown ethers    -   Polycarboxylates such as citrates, oxalates, tartrates,        succinates, and malonates;    -   Phosphonates;    -   Polyamines with a varied number and identity of ligands in the        chain.        -   Tetradentate:    -   2,3,2-triethylenetetramine [N_(H2)CH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂NH₂]    -   3,2,3-triethylenetetramine [NH₂CH₂CH₂CH₂NHCH₂CH₂NHCH₂CH₂CH₂NH₂]    -   3,3,3-triethylenetetramine        [NH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂NH₂]        -   NSSN-Type Ligands:    -   NH₂CH₂CH₂SCH₂CH₂SCH₂CH₂NH₂    -   NH₂CH₂CH₂SCH₂CH₂SCH₂CH₂NH₂    -   NH₂CH₂CH₂SCH₂CH₂CH₂SCH₂CH₂NH₂    -   NH₂CH₂CH₂CH₂SCH₂CH₂SCH₂CH₂CH₂NH₂        -   SNNS-Type Ligands:    -   HSCH₂CH₂N(CH₃)CH₂CH₂N(CH₃)CH₂CH₂SH    -   HSCH₂CH₂N(CH₃)CH(CH₃)CH₂N(CH₃)CH₂CH₂SH    -   HSCH₂CH₂N(CH₃)CH₂CH₂CH₂N(CH₃)CH₂CH₂SH    -   HSC(CH₃)2CH₂NHCH₂CH₂NHCH₂C(CH₃)2CSH        -   Pentadentate:    -   NH₂CH₂CH₂SCH₂CH₂N(CH₃)CH₂CH₂SCH₂CH₂NH₂    -   NH₂CH₂CH₂SCH₂CH₂SCH₂CH₂SCH₂CH₂NH₂.        Method of the Invention

The present invention also provides a method in which the sensordescribed above is used in for detecting an analyte. In accordance withthis method, the sample is applied to the sensor. A potential is appliedbetween the working and counter electrodes, and the current between theelectrodes is observed. An indication of the presence or absence ofdetectable amounts of analyte in the sample is generated based on theobserved current. As will be appreciated by persons skilled in the art,this method is advantageously practices using a meter that contains theelectronics for applying the potential, observing the current, andgenerating a determination of analyte and conveying it to the user.Numerous meters for this purpose have been disclosed, and the particulardesign of the meter is not critical.

In accordance with a first embodiment of the method of the invention,the voltage difference applied to electrodes. V_(app), is greater thanboth E⁰ _(shut) and E⁰ _(med) such that both the shuttle and themediator can react at the electrodes as depicted in FIG. 3.

In accordance with a second embodiment of the method of the invention,E⁰ _(shut) is less than E⁰ _(med) and the voltage difference applied toelectrodes, V_(app), is intermediate between E⁰ _(shut) and E⁰ _(med)such that only the shuttle reacts at the electrodes as depicted in FIG.7.

EXAMPLE 1

Calibration curves were prepared to compare the performance of a reagentformulae that had ferricyanide alone as the mediator, and one inaccordance with the invention that use ferricyanide as a shuttle and thepoorly soluble PF₆ salt of [Os(MeBpy)₂(Im)₂]^(2+/3+) as a mediator forthe reaction of glucose and glucose oxidase. In each case, the amount ofglucose oxidase was the same, and the applied voltage was 150 mV. Thetwo reagent formulations were as follows:

1. Ferricyanide alone, as both shuttle and mediator: reagent: 100 mMferri, 5 mM phosphate buffer pH 8.1, +stabilizers, sample: 200 mMcitrate buffer pH 4.1, 133 nK NaCl

2. Ferricyanide (as a shuttle)+Osmium salt (as efficient mediator):reagent: saturated (<1 mM) Osmium salt, 100 mM ferri, 5 mM phosphatebuffer pH 8.1, and stabilizers, sample 50 mM phosphate buffer pH 8.1,133 mM NaCl.

In each case, the stabilizers were polyethyleneglycol and erythritol.Sample pH is different in each case. -The chosen pH represents theoptimal pH for that mediator system. One would not expect Ferricyanide(which is optimized at pH 4.1) to mediate well at pH 8.1, whereas it isideal for Osmium, and the difference in pH was therefore adopted toconfirm that better performance was in fact being achieved and that theobserved difference was not an artifact.

FIG. 9 shows the observed calibration curves (current versus glucoseconcentration) for the two reagents. When ferricyanide is used alone, ithas to function as a mediator and a shuttle and is therefore inefficientresulting in lower currents at any given concentration. When used incombination, ferricyanide (high solubility, slow electron transfer ratefrom enzyme) and osmium (low solubility, fast rate of electron transferfrom the reduced enzyme) function as an efficient shuttle and mediatorrespectively, resulting in higher observed currents and a greater slope.Higher observed currents and the greater slope allows for improvedaccuracy and greater dynamic range.

EXAMPLE 2

A reagent formulation was prepared as follows:

100 mM phosphate buffer (pH 7.0), 100 mM ferricyanide, 50 mN alanineanhydride, 1 mM Os(Mebpy)₂PicCl (˜1 mg/ml), 2.6 mg/ml Surfactant 10 G(Arch Chemicals).

Good results measuring glucose were obtained using this formulation.

EXAMPLE 3

A reagent formulation was prepared as follows:

100 mM phosphate buffer (pH 7.0), 100 mM ferricyanide, 50 mN alanineanhydride, saturated (<1 mM) [Os(Mebpy)₂Im₂](PF₆)₂, 2.6 mg/ml Surfactant10 G (Arch Chemicals), 1% w/v silica (AERODISP W7520N, Degussa).

Good results measuring glucose were obtained using this formulation.

In the formulas above, MeBpy=4.4′ dimethyl-2,2′-bipyridyl,Pic=Picolinate, the conjugate base of Picolinic Acid. Alanine Anhydrideis a stabilizer (See U.S. Pat. No. 3,243,356) and silica is a stabilizer(See, U.S. Pat. No. 3,556,945).

1. A method for detecting glucose in a sample comprising the steps of:(a) applying a sample to be evaluated for the presence of glucose to atest strip, wherein the test strip comprises: a support portion defininga sample chamber; a working and a counter electrode disposed within thesample chamber; and a reagent composition disposed in the samplechamber, said reagent composition comprising (i) an active redox enzymeeffective to oxidize glucose in the sample, (ii) a mediator compound,and (iii) a shuttle compound, wherein the shuttle compound and themediator compound are different from one another; wherein when a samplecontaining analyte is introduced into the sample chamber, the redoxenzyme oxidizes glucose the analyte and produces an inactive enzyme thatis reduced, the mediator is [Os(Mebpy)₂Pic]^(+/2+) and the shuttle isferri/ferrocyanide, and (b) applying a potential between the working andthe counter electrodes to generate current that is indicative of theamount of glucose in the sample.
 2. A method for detecting an analyte ina sample comprising the steps of: introducing the sample into a sensor,wherein said sensor comprises: (a) a support portion defining a samplechamber; (b) a working and a counter electrode disposed within thesample chamber; and (c) a reagent composition disposed in the samplechamber, said reagent composition comprising (i) an active redox enzymeeffective to oxidize the analyte, (ii) a mediator compound, and (iii) ashuttle compound, wherein when a sample containing analyte is introducedinto the sample chamber, the redox enzyme oxidizes the analyte andproduces an inactive enzyme that is reduced, the mediator is[Os(Mebpy)₂Pic]^(+/2+) and the shuttle is ferri/ferrocyanide.
 3. Themethod of claim 2, wherein the shuttle compound is present in at least aten-fold excess with respect to the mediator compound on a molar basis.4. The method of claim 2, wherein the reagent composition furthercomprises a third electron transfer agent that transfers electronsbetween the mediator and the shuttle.
 5. The method of claim 2, whereinthe analyte is glucose and the enzyme is glucose oxidase.
 6. A sensorfor the detection of an analyte in a sample, comprising: (a) a supportportion defining a sample chamber; (b) a working and a counter electrodedisposed within the sample chamber; and (c) a reagent compositiondisposed in the sample chamber, said reagent composition comprising (i)an active redox enzyme effective to oxidize the analyte, (ii) a mediatorcompound, and (iii) a shuttle compound, wherein when a sample containinganalyte is introduced into the sample chamber, the redox enzyme oxidizesthe analyte and produces an inactive enzyme that is reduced, themediator is [Os(Mebpy)₂Pic]^(+/2+) and the shuttle isferri/ferrocyanide.
 7. The sensor of claim 6, wherein the shuttlecompound is present in at least a ten-fold excess with respect to themediator compound on a molar basis.
 8. The sensor of claim 6, whereinthe reagent composition further comprises a third electron transferagent that transfers electrons between the mediator and the shuttle. 9.The sensor of claim 6, wherein the analyte is glucose and the enzyme isglucose oxidase.